IMPDH inhibition activates TLR‐VCAM1 pathway and suppresses the development of MLL‐fusion leukemia

Abstract Inosine monophosphate dehydrogenase (IMPDH) is a rate‐limiting enzyme in de novo guanine nucleotide synthesis pathway. Although IMPDH inhibitors are widely used as effective immunosuppressants, their antitumor effects have not been proven in the clinical setting. Here, we found that acute myeloid leukemias (AMLs) with MLL‐fusions are susceptible to IMPDH inhibitors in vitro. We also showed that alternate‐day administration of IMPDH inhibitors suppressed the development of MLL‐AF9‐driven AML in vivo without having a devastating effect on immune function. Mechanistically, IMPDH inhibition induced overactivation of Toll‐like receptor (TLR)‐TRAF6‐NF‐κB signaling and upregulation of an adhesion molecule VCAM1, which contribute to the antileukemia effect of IMPDH inhibitors. Consequently, combined treatment with IMPDH inhibitors and the TLR1/2 agonist effectively inhibited the development of MLL‐fusion AML. These findings provide a rational basis for clinical testing of IMPDH inhibitors against MLL‐fusion AMLs and potentially other aggressive tumors with active TLR signaling.


Introduction
MLL-fusion leukemias are aggressive forms of acute leukemias carrying chimeric fusion of the MLL (also called KMT2A) gene. The MLL gene encodes a histone H3 lysine 4 (H3K4) methyltransferase and is the frequent target of chromosomal translocations, resulting in the fusion of the 5 0 portion of MLL to a number of different partner genes (Yokoyama, 2015;Slany, 2016). MLL-fusions, such as MLL-AF9, MLL-AF4, and MLL-ENL, are recurrently found in a subset of acute myeloid leukemia (AML) and B-cell precursor acute lymphoblastic leukemia (B-ALL). The MLL-fusion proteins lose the H3K4 methyltransferase activity but instead acquire aberrant functions for epigenetic regulation. Despite recent advances in understanding molecular pathogenesis and therapeutic approaches, many patients with MLL-fusion leukemia still have poor outcomes.
Studies have identified crucial factors that are involved in MLLfusion driven leukemogenesis, including interacting proteins (Yokoyama et al, 2005;Yokoyama & Cleary, 2008), epigenetic regulators (Okada et al, 2005;Zuber et al, 2011b;Harris et al, 2012;Neff et al, 2012;Asada et al, 2018), transcription factors (Zuber et al, 2011a;Ye et al, 2015), and signaling molecules (Wang et al, 2008(Wang et al, , 2010Kuo et al, 2013). In AML, MLL rearrangements are most strongly associated with French-American-British subtypes M4/M5 that have monocytic/macrophage-like characteristics (Tien et al, 2000). Because Toll-like receptors (TLRs) constitute a key signaling system in these innate immune cells (Takeda & Akira, 2005), the TLR pathways could also be therapeutic targets in MLL-fusion AMLs. Toll-like receptors mainly transduce signals through an adaptor protein MyD88. MyD88 forms the Myddosome with IRAK kinases, which induces association and activation of the E3 ubiquitin ligase TRAF6. TRAF6 promotes K63-linked polyubiquitination of the protein kinase TAK1, and TAK1 then activates NF-jB and MAPK pathways. The activated NF-jB induces expression of pro-inflammatory cytokines and adhesion molecules, such as TNFa, IL-6, and Vcam1. The active IRAK-NF-jB pathway has been shown to be necessary to maintain aberrant epigenetic programs induced by MLL-fusion proteins (Goyama & Mulloy, 2013;Kuo et al, 2013;Liang et al, 2017). Importantly, excessive activation of the TLR-NF-jB pathway could also prevent tumor development. Indeed, studies have shown that agonistic targeting TLR1/2 or TLR8 induced apoptosis and differentiation of MLL-fusion AMLs (Ignatz-Hoover et al, 2015;Eriksson et al, 2017).
Uncontrolled cell proliferation is a hallmark of tumors, including MLL-fusion leukemia, which requires adequate nucleotide biosynthesis. Guanine nucleotides play essential roles in diverse biological processes and are synthesized de novo or recycled via salvage pathways. Inosine monophosphate dehydrogenase (IMPDH) is a ratelimiting enzyme in de novo guanine nucleotide synthesis pathway, which is responsible for the conversion of IMP to XMP (Hedstrom, 2009;Naffouje et al, 2019;Fig 1A). In mammals, there are two IMPDH isotypes: IMPDH1 and IMPDH2. IMPDH2 is a predominant isotype in most tissues, especially in tumors and highly replicative cells (Collart et al, 1992). Several IMPDH inhibitors, such as Mycophenolic acid (MPA) and mizoribine, are widely used as immunosuppressants for the prevention of organ transplant rejection (Naffouje et al, 2019). In addition, recent studies have shown the antitumor activity of IMPDH inhibition against a variety of tumors, such as glioblastoma , ASCL1-low-smallcell lung cancer (Huang et al, 2018), relapsed ALL with NT5C2 mutations (Tzoneva et al, 2018) and AML cell lines (Murase et al, 2016;Yang et al, 2017). In clinical trials, tiazofurin and FF-10501-01 (a prodrug of mizoribine) showed clinical activity in patients with myeloid neoplasms (Tricot et al, 1989;Garcia-Manero et al, 2020). However, the clinical trial using FF-10501-01 was discontinued due to increased mucositis events (Garcia-Manero et al, 2020), indicating that further optimization of dose schedule is required to minimize side effects of IMPDH inhibitors. Another concern for the use of IMPDH inhibitors as anticancer drugs is their immunosuppressive activity. Given the importance of tumor immunosurveillance, the therapeutic effects of IMPDH inhibitors could be attenuated in vivo.
In this study, we showed the potent antileukemia effect of two IMPDH inhibitors, MPA and FF-10501-01, on MLL-fusion AMLs. Importantly, we found that alternate-day administration of IMPDH inhibitors to mice suppressed the development of MLL-AF9-driven AML in vivo without substantially reducing the number of immune cells. Our study also revealed the unexpected effect of IMPDH inhibitors to induce overactivation of the TLR signaling and upregulation of VCAM1 in AML cells, which is likely to contribute to their antileukemia effects.

A
Schematic of de novo purine nucleotide synthesis. B, C IC 50 values for MPA-response curves using cell viability assay were shown. Human AML cell lines (with MLL-fusions: MV4;11, MOLM13, NOMO1 and THP1, without MLL-fusions: HL60, Kasumi-1, OCI-AML3 and U937), human cord blood (CB) CD34 + cells, and those expressing MLL-AF9 (CB-MLL-AF9#1, 2, 3) or MLL-ENL (CB#MLL-ENL-1, 2), and patient cells with or without MLL-fusions (AML#1, 2, 3 or AML#4, 5, 6) were treated with titrating doses of MPA (  and B), indicating that expression of IMPDH1/2 did not predict sensitivity of each PDX-AML to MPA. Another IMPDH inhibitor FF-10501-01 also showed the growth-inhibitory effect on MLL-AF9expressing CB cells, while it showed only modest effect on normal CB cells ( Fig 1D). Mechanistically, MPA treatment caused a cell cycle arrest at G0/G1-phase and triggered apoptosis in MLL-AF9expressing CB cells, MV4;11 cells and MOLM13 cells (Fig 1E; Appendix Fig S2A and B). Furthermore, MPA-induced differentiation of MLL-AF9-expressing CB cells, as evidenced by the changes in surface marker expression or morphological changes (Fig 1F and  G). These MPA-induced changes in MLL-fusion leukemia cells were reversed by the supplementation of guanosine (Fig 1E-G; Appendix Fig S1), confirming that guanine nucleotide depletion underlies the effects of MPA. We also found that MPA synergized with a BCL2 inhibitor venetoclax, but not with a hypomethylating agent decitabine, to inhibit the growth of MLL-AF9-expressing CB cells (Appendix Fig S3A and B). We then assessed the role of IMPDH1 and IMPDH2 in MLL-fusion leukemias using the CRISPR/ Cas9 system. Genetic depletion of IMPDH2 inhibited the growth of MOLM13 cells and MLL-ENL-expressing CB cells, while IMPDH1 depletion showed little effects on their growth. (Figs 1H and I,and EV2C and D). These data indicate that IMPDH2 is a key molecule in MLL-fusion leukemia. Thus, IMPDH inhibition suppresses cell cycle progression, induces apoptosis, and enhances differentiation in MLL-fusion AMLs, thereby inhibits their growth. Because MLL-fusions are also known to drive the development of B-ALL, we then assessed the effect of MPA on CB cells expressing MLL-Af4 that recapitulate t (4;11) pro-B ALL  using a coculture assay with murine stromal cell line MS-5 (Appendix Fig S4A). Addition of MPA induced a dramatic decrease in the formation of leukemic cobblestone-forming cells with a concomitant increase of CD10 + expression in MLL-Af4 cells in a concentration-dependent manner (Appendix Fig S4B and C). Furthermore, MPA inhibited the cobblestone formation of PDX cells derived from two B-ALL patients (ALL#1 and ALL#2) at relatively low concentration (1 lM). The MPA-induced inhibition of cobblestones formation of ALL#1 cells was partially reversed by guanosine supplementation (Appendix Table S1 and Appendix Fig S4D and E). Thus, IMPDH inhibitors show robust growth-inhibitory effects on leukemias with MLL-fusions in vitro.

IMPDH inhibitors suppress the development of MLL-AF9-driven AML without eradicating immune cells in mice
We next assessed the in vivo effect of IMPDH inhibitors using a mouse AML model driven by MLL-AF9. Mouse bone marrow (BM) progenitors were transduced with MLL-AF9 (co-expressing GFP) and were transplanted into C57BL/6J recipient mice. MLL-AF9expressing AML cells were collected from moribund mice and used for serial transplantation. The mice that received MLL-AF9 cells were then treated with vehicle or IMPDH inhibitors every other day from Day 1 (Fig 2A). Treatment with both mycophenolate mofetil (MMF; a prodrug of MPA, 120 mg/kg) and FF-10501-01 (160 mg/ kg) inhibited the engraftment of MLL-AF9 cells and significantly prolonged survival of these mice (Fig 2B and C). Delayed initiation of FF-10501-01 (from Day 12 to Day 23) still prolonged the survival of MLL-AF9 leukemia mice, with reduction of GFP + leukemia cells in peripheral blood (Fig EV3A and B). To assess the mechanisms of drug action in vivo, we then treated the mice with IMPDH inhibitors for two consecutive days and analyzed the bone marrow cells on the next day ( Fig 2D). We found that FF-10501-01 induced myeloid differentiation of MLL-AF9 cells in vivo, as evidenced by the characteristic morphological changes (Fig 2E), the increase in Mac-1 + Gr-1 + cells, and the decrease in c-Kit + Gr-1 À cells ( Fig 2F). By contrast, FF-10501-01 did not induce cell cycle arrest and apoptosis in MLL-AF9 cells in this experimental condition ( Fig 2G).
To assess the effect of IMPDH inhibition on human AML cells in vivo, we transplanted the MPA-sensitive PDX cells with MLLfusions (Appendix Table S1) to the immunodeficient NOD/RAG1/ 2 À/-IL2Rc À/À (NRGS) mice expressing human SCF, IL-3, and GM-CSF (Barve et al, 2018), and treated them with IMPDH inhibitors (MMF or FF-10501-01) for 3 weeks ( Fig 2H). Although both inhibitors showed a weak tendency to reduce the engraftment of human AML cells in bone marrow, neither of them improved the survival ▸ Figure 2. IMPDH inhibitors suppress the growth of MLL-fusion leukemia in vivo.
A Experimental scheme used in (B and C). C57BL/6J mice were transplanted with MLL-AF9-GFP cells and were treated with vehicle or MMF or FF-10501-01. B Kaplan-Meier survival curves of MLL-AF9 leukemia mice treated with vehicle or MMF (n = 12 per group). Frequencies of GFP + leukemic cells in peripheral blood at day10 are also shown (mean AE SEM) on the right side (vehicle: n = 3, MMF: n = 4). Unpaired Student's t-test (two-tailed) were used for pairwise comparisons of significance. C Kaplan-Meier survival curves with vehicle (n = 5), FF-10501-01 (n = 10). Frequencies of GFP + leukemic cells in peripheral blood at day10 are also shown (mean AE SEM) on the right side (vehicle: n = 5, FF-10501-01: n = 5). Unpaired Student's t-test (two-tailed) were used for pairwise comparisons of significance. D Experimental scheme used in (E-G). C57BL/6J mice were transplanted with MLL-AF9-GFP cells and were treated with vehicle or FF-10501-01 on day 11 and 12. GFP + MLL-AF9 leukemia cells collected from mice were analyzed on day 13. E Wright-Giemsa staining of MLL-AF9 cells (Scale bar: 20 lm). F Representative FCM plots of frequencies of c-Kit + Gr-1 À and Mac-1 + Gr-1 + in leukemic cells and their quantification of independent mice (n = 10 per group). Unpaired Student's t-test (two-tailed) were used for pairwise comparisons of significance. G Cell-cycle status (left) and apoptosis (right) were assessed. Frequencies of S/G2/M phase cells and Annexin V + cells in GFP + MLL-AF9 leukemia cells (n = 10 per group).
Data information: All data are shown as mean AE SEM. Log-rank test was used to compare the survival curves.  Fig S5A and B). These data suggest that the lack of an intact immune system in NRGS mice may limit the therapeutic effect of IMPDH inhibitors in this PDX model. Indeed, we found that FF-10501-01 treatment provided more survival benefits in immunocompetent C57BL/6J mice than immunodeficient NOD/SCID-IL2Rc À/À (NSG) mice even in the mouse MLL-AF9 model (Fig 2K), indicating that systemic immune responses contribute to the in vivo effect of IMPDH inhibition. Because IMPDH inhibitors are known to act as immunosuppressants, we next assessed whether our dosing schedule for FF-10501-01 affects the numbers of immune cells in leukemic mice. We treated the MLL-AF9-bearing mice with FF-10501-01 every other day from Day 1 to Day 9 and then examined the numbers and frequency of immune cells in bone marrow and spleen at Day 10. In agreement with earlier results, treatment with FF-10501-01 substantially inhibited the growth of MLL-AF9 cells. Although FF-10501-01 treatment modestly decreased the total numbers of normal hematopoietic cells (Appendix Fig S6A and B), it did not reduce the frequency of B and T lymphocytes, NK cells, macrophages, and neutrophils in the bone marrow and spleen (Appendix Fig S6C and D). Thus, our alternateday treatment with IMPDH inhibitors did not eradicate immune cells in mice, and the remaining immune cells are likely to enhance the antileukemia effect of IMPDH inhibitors in vivo.
Taken together, these results suggest that IMPDH inhibitors suppress the in vivo development of MLL-AF9-driven AML with the assistance of immune cells.

p53-p21 activation is dispensable for the antileukemia effect of IMPDH inhibitors
Several studies have proposed that activation of the p53-p21 pathway contributes to the antitumor effect of IMPDH inhibitors (Messina et al, 2004;Sun et al, 2008). Indeed, we observed consistent upregulation of p53 in CB cells, RUNX1-ETO-expressing CB cells (preleukemic cells; Goyama et al, 2016), and two MLL-AF9expressing CB cells treated with MPA. The expression of p21 was also upregulated in most of these cells, except for an MLL-AF9 leukemia clone, CB-MLL-AF9#1 ( Fig 3A). The p53-p21 upregulation was reversed by simultaneous addition of guanosine, indicating that this was an on-target effect of MPA. To examine the role of p21 in the regulation of MPA's efficacy, we depleted p21 in CB-MLL-AF9#2 using a p21-targeting shRNA (10; Fig 3B). MPA inhibited the growth of p21-depleted MLL-AF9 cells as efficiently as control cells ( Fig 3C). These data, together with the fact that CB-MLL-AF9#1 lacking p21 expression is sensitive to MPA, indicate that p21 is dispensable for the growth-inhibitory effect of IMPDH inhibition.
We next examined whether p53 activation is necessary for the antileukemia effect of MPA. We transduced vector or a dominantnegative p53 fragment (p53DD) into MLL-AF9-expressing CB cells ( Fig 3D) and assessed the effect of MPA on these cells in vitro. MLL-AF9 cells transduced with p53DD were equally sensitive to MPA relative to control cells ( Fig 3E). Finally, we assessed the sensitivity of p53-deficient mouse MLL-AF9 leukemia cells to IMPDH inhibition in vivo. We generated p53-deficient leukemia cells by expressing MLL-AF9 into bone marrow progenitors derived from p53 knockout mice . The p53-deficient MLL-AF9 cells were transplanted into recipient mice, and the mice were treated with vehicle or IMPDH inhibitors. The p53-MDM2 interaction inhibitor DS-5272  was also used as a control ( Fig 3G). Consistent with the in vitro data, treatment with FF-10501-01 induced p53 upregulation in MLL-AF9 cells in vivo, which was not observed in p53-deficient MLL-AF9 cells ( Fig 3F). Again, we found that p53-deficient MLL-AF9 cells were still sensitive to FF-10501-01 treatment in vivo while resistant to DS-5272 ( Fig 3H). Taken together, we concluded that activation of the p53-p21 pathway is not essential for the antileukemia efficacy of IMPDH inhibitors.

IMPDH inhibition induces inflammation and alters metabolic homeostasis in vivo
To assess the molecular changes induced by IMPDH inhibition in MLL-AF9 cells, we first examined expression profiles of vehicle-or FF-10501-01-treated MLL-AF9 cells after two consecutive days of treatment in vivo ( Fig 4A). A clear separation of FF-10501-01treated cells and controls was observed ( A Immunoblotting of the cord blood cells and those expressing RUNX1-ETO or MLL-AF9. The cells were incubated with/without 1 lM MPA (M) and 100 lM Guanosine (G) for 24 h. B CB-MLL-AF9#2 cells were transduced with a Non-targeting (NT) control and an shRNA targeting p21 (sh-p21). Cells were incubated with/without 1 lM MPA (M) and 100 lM guanosine (G) for 24 h. Total cell lysates were analyzed by western blotting using the antibodies for p21 and Tubulin. C CB-MLL-AF9#2 cells transduced with NT or sh-p21 shRNAs were incubated with MPA at the indicated concentration for 72 h. Cell viability assays were performed using WST-1 in three technical replicates. D CB-MLL-AF9#2 cells were transduced by vector control or p53DD (dominant-negative) and were incubated with/without 1 lM MPA (M) and 100 lM guanosine (G) for 24 h. Total cell lysates were analyzed by western blotting using the antibodies for p53 and Tubulin. Note the elevated levels of endogenous p53 protein (upper arrow) in p53DD (lower arrow)-expressing cells. E Cells were incubated with MPA at the indicated concentration with/without 100 lM guanosine for 72 h then measured with WST-1 in three technical replicates. F Mouse bone marrow c-Kit + cells derived from Trp53 À/À mice were transduced with MLL-AF9-GFP and were transplanted into mice to generate p53-deficient (p53 À/À ) leukemia cells. GFP + MLL-AF9 leukemia cells were collected from bone marrows of vehicle-or FF-10501-01-treated wild-type and p53 À/À leukemic mice. Expression levels of p53 and Tubulin were assessed by western blotting 24 h after the treatment. G Experimental scheme used in (H). The p53-deficient MLL-AF9-bearing mice were treated with vehicle, FF-10501-01 or DS-5272 every other day from day 1. H Kaplan-Meier survival curves of p53 À/À MLL-AF9 leukemia mice treated with vehicle or FF-10501-01 or DS-5272 (n = 6 per group). Statistical significance was evaluated by the log-rank test (left). Frequency of GFP + leukemia cells in peripheral blood at day 19 (right, n = 6 per group). A two-tailed unpaired t-test was used for the comparison.
Data information: All data are shown as mean AE SEM. Source data are available online for this figure. et al, 2005) revealed upregulation of inflammation-and interferonassociated genes, suggesting that IMPDH inhibition triggers an immune-inflammatory response in MLL-AF9 cells ( Fig 4D). IMPDH inhibition also induced downregulation of mTORC1 signaling ( Fig 4E), which is consistent with a previous report (Emmanuel et al, 2017). Among the mTORC1 pathway molecules, we found that SLC7A5 and SLC3A2 were downregulated in FF-10501-01-treated MLL-AF9 cells ( Fig 4F). SLC7A5 and SLC3A2 encode CD98 and Lat1, respectively, and they form a heterodimeric membrane complex that is involved in the uptake of essential amino acids and adhesive signals (Feral et al, 2005;Hafliger & Charles, 2019). We confirmed reduced surface expression of CD98/Lat1 in mouse MLL-AF9 cells in vivo (Fig 4G), and the reduction was also confirmed in human MOLM13 cells and MV4;11 cells treated with FF-10501-01 in vitro ( Fig EV4). Given the important role of CD98 in AML progression (Bajaj et al, 2016), the CD98/Lat1 downregulation may contribute to the therapeutic effect of IMPDH inhibitors on AML. By contrast, FF-10501-01 treatment did not affect the expression of MLL-AF9 target genes, such as HoxA9 and Meis1 (Fig 4H), indicating the little effect of IMPDH inhibition on the aberrant epigenetic programs induced by MLL-AF9. We then performed metabolomic analyses using MLL-AF9 cells collected from leukemic mice treated with vehicle or FF-10501-01. As expected, FF-10501-01 treatment tended to reduce intracellular levels of guanine nucleotides in MLL-AF9 cells, although the levels of GDP/GTP were not significantly downregulated probably due to the in vivo compensatory mechanisms ( Fig 4I). Interestingly, levels of other nucleoside monophosphate (AMP, CMP, and UMP) were also downregulated upon FF-10501-01 treatment, indicating the presence of crosstalk between each nucleotide (Appendix Fig S7). We also found reduced levels of several essential amino acids (EAAs), including valine, isoleucine, leucine, tyrosine, and phenylalanine in FF-10501-01-treated MLL-AF9 cells ( Fig 4J). Given that Lat1 preferentially transports these amino acids, the Lat1 downregulation upon IMPDH inhibition may be responsible for the reduction in EAAs. In addition, FF-10501-01 treatment of MLL-AF9 cells resulted in a wide range of alterations in the levels of bases and amino acids (Appendix Fig S8). Thus, IMPDH inhibition in vivo provokes inflammatory responses and metabolic alterations in MLL-AF9 AML cells.

IMPDH inhibition induces overactivation of TLR signaling in AML cells
Previous reports have shown that IMPDH suppresses TLR2mediated NF-jB activation (Toubiana et al, 2011) and TLR-7mediated antiviral activity (Lee et al, 2006). Therefore, we speculated that activation of TLR signaling could underlie the increased inflammation in MLL-AF9 cells treated with IMPDH inhibitors. To test this hypothesis, we first assessed the effect of MPA on TLR signaling pathways using a mouse pro-B cell line Ba/F3 that expresses individual TLRs and the NF-jB -GFP reporter (Matsumoto et al, 2008;Shibata et al, 2012;Sato et al, 2017; Fig 5A). The addition of MPA increased TLR2-, TLR4-, TLR5-, and TLR7/8-mediated activation of NF-jB in a dose-dependent manner (Fig 5B), indicating the inhibitory role of IMPDH in the TLR-NF-jB pathway. Next, we assessed the role of IMPDH in the regulation of TRAF6, an E3 ubiquitin ligase that plays a pivotal role in linking TLR signaling to NF-jB. We introduced FLAG-tagged TRAF6 and HA-tagged K63ubiquitin into 293 T cells. After 24 h, these cells were treated with MPA with/without guanosine. Cell lysates were subjected to immunoprecipitation with FLAG antibody for TRAF6, followed by immunoblotting with anti-HA to detect ubiquitinated TRAF6. We found that MPA treatment increased TRAF6 autoubiquitination (an active marker of TRAF6), which was reversed by the supplementation of guanosine ( Fig 5C). Thus, IMPDH inhibition induces activation of the TLR-TRAF6-NF-jB signaling pathway.
◀ Figure 4. The effect of IMPDH inhibition on gene expression and metabolic profiles.

A
Experimental scheme used in (B-J). C57BL/6J mice were transplanted with MLL-AF9-GFP cells and were treated with vehicle or FF-10501-01 on day 11 and 12. GFP + MLL-AF9 leukemia cells collected from mice were used for RNA-seq and metabolome analysis at day 13. B, C Principal Component Analysis (B) and hierarchical clustering (C) of RNA-Seq data. n = 3 per group. D, E GSEA for up-(D) and down-(E) regulated genes in FF-10501-01-treated MLL-AF9 cells using the Hallmark collections of the GSEA MSigDB (http://software. broadinstitute.org/gsea/msigdb). The x-axis shows the P-value (Àlog10). F mRNA expression (FPKM) of SLC7A5 and SLC3A2 in vehicle-or FF-10501-01-treated leukemia cells. The P-value was calculated by Cuffdiff. n = 3 (biological replicates) for each group. G MFI of CD98 on the vehicle-or FF-10501-01-treated leukemia cells in mice. A two-tailed unpaired t-test was used for the comparison. n = 3 (biological replicates) for each group. H mRNA expression (FPKM) of HoxA9 and Meis1 in vehicle-or FF-10501-01-treated leukemic cells. P-value was calculated by Cuffdiff. n = 3 (biological replicates) for each group. I, J Vehicle-or FF-10501-01-treated leukemic cells were used for metabolome analyses. n = 3 (biological replicates) per group. Levels of guanine nucleotides (GMP, GDP, and GTP) (I) and amino acid contents (J) are shown. Data are presented as mean AE SEM (I) or as a viridis heatmap (Plot is SEM) (J). A two-tailed unpaired ttest was used for the comparison.
Data information: All data are shown as mean AE SEM.
A Experimental scheme used in (B). Ba/F3 cells were transduced with NF-jB-GFP reporter and were then transduced with individual mouse TLR4, TLR5, or TLR7 to establish BajB cells expressing each TLR. TLR2 is expressed in parent Ba/F3 cells. B The TLR-expressing BajB cells were stimulated with the corresponding TLR ligands (100 ng/ml Pam3CSK4 for TLR2, 100 ng/ml LPS for TLR4, 10 ng/ml Flagellin for TLR5, 100 ng/ml R848 for TLR7) with titrating doses of MPA (1-10 lM). GFP expression was assessed 24 h after the stimulation. C 293 T cells were transfected with FLAG-tagged TRAF6 and HA-tagged K63-ubiquitin. After 24 h, these cells were treated with 3 or 10 lM MPA (M-3 lM or M-10 lM) with/without 100 lM guanosine (G). IRAK1/4 inhibitor (IRAK1/4 i) was also used as a control. Whole-cell extracts were immunoprecipitated with anti-FLAG antibody, and ubiquitinated TRAF6 was detected with anti-HA antibody. D, E Murine bone marrow-derived macrophages (BMDMs) were treated with vehicle, 10 lM MPA alone (D) or co-treated with vehicle, 10 lM MPA (M), 100 ng/ml Pam3CSK4 and 100 lM Guanosine (G) for 4 h (E), as indicated. N = 3 (technical replicates) per group. mRNA levels of TNF-a and IL-1b were measured by qPCR. Two-tailed unpaired t-tests were used for the comparison in (D). Ordinary one-way ANOVA was used for the comparison in (E). F, G (F) THP1 cells and (G) MLL-AF9-expressing CB cells (CB-MLL-AF9#1) were pretreated with 10 lM MPA for 2 h and then treated with 1,000 ng/ml Pam3CSK4 for 0, 30, 60, and 120 min. Cells were lysed and subjected to SDS/PAGE. Levels of total IjBa, phosphate-p38, total p38, and GAPDH were evaluated by western blotting.
Data information: All data are shown as mean AE SEM. Source data are available online for this figure. We then examined the effect of MPA on induction of proinflammatory cytokines. Mouse bone marrow-derived macrophages (BMDMs) were treated with MPA, guanosine, and the TLR1/2 agonist Pam3CSK4 for 4 h, and levels of TNF-a and IL-1b were assessed. Mycophenolic acid treatment modestly increased TNF-a and IL-1b expression ( Fig 5D) and enhanced Pam3CSK4-induced upregulation of these pro-inflammatory cytokines in BMDMs ( Fig 5E). The collaborative effect of MPA and Pam3CSK4 was abrogated with the supplementation of guanosine, indicating that the reduction in guanine nucleotides triggered the inflammatory responses ( Fig 5E). We also found that MPA treatment increased IjBa degradation and p38 phosphorylation induced by Pam3CSK4 in THP1 cells and CB cells expressing MLL-AF9 (Fig 5F and G). Collectively, these data suggest that IMPDH inhibition in fact promotes TLR signaling, which induces excessive inflammation in AML cells.

Vcam1 regulates cell-cell interaction and suppresses proliferation in MLL-AF9 cells
To further assess the molecular alterations induced by IMPDH inhibition, we next performed single-cell mass cytometry analyses using MLL-AF9 cells collected from mice treated with vehicle or FF-10501-01( Fig 6A). Four populations were identified according to Mac-1, Gr-1, and c-Kit expression on the Spanning-tree Progression Analysis of Density-normalized Events (SPADE) tree (Fig 6B;  Appendix Fig S9A and B). We found that FF-10501-01 induced Vcam1 upregulation in Mac-1 + fraction, phospho-STAT1 upregulation in Mac-1 + c-Kit + fraction, CXCR4 downregulation in c-Kit + fraction and HIF-1a upregulation in Gr-1 + fraction in MLL-AF9 cells (Fig 6C; Appendix Fig S9C). Flow cytometry analysis also confirmed the Vcam1 upregulation in FF-10501-01-treated leukemia cells (Fig 6D). Guanosine supplementation reversed the FF-10501-01induced Vcam1 upregulation only partially (Appendix Fig S10A), indicating that Vcam1 upregulation in MLL-AF9 cells was induced by both direct and indirect (probably related to enhanced inflammation and myeloid differentiation) effects of IMPDH inhibition.
Because Vcam1 is known to be activated by pro-inflammatory cytokines (Kong et al, 2018), the overactive TLR signaling in FF-10501-01-treated MLL-AF9 cells could also contribute to Vcam1 upregulation. To assess the role of Vcam1 in the development of MLL-AF9 leukemia, we then transduced Cas9 together with a vector or two independent Vcam1-targeting single guide RNAs (sgRNAs) into mouse MLL-AF9 cells. Vcam1-targeting sgRNAs induced nearly complete depletion of Vcam1 on the cell surface of MLL-AF9 cells. Interestingly, Vcam1-depleted MLL-AF9 cells lost cell-cell contacts and stopped forming cell aggregates in suspension cultures (Fig 6E). Conversely, Vcam1 overexpression in MLL-AF9 cells using SunTag system (Tanenbaum et al, 2014) markedly increased the formation of cell aggregation in culture (Fig 6G). We also found that Vcam1 depletion promoted cell cycle progression (Fig 6F), whereas Vcam1 overexpression inhibited it in MLL-AF9 cells (Fig 6H). Depletion of Vcam1 showed little effects on FF-10501-01-induced myeloid differentiation in MLL-AF9 cells (Appendix Fig S10B).
To determine the role of Vcam1 in AML progression in vivo, we next transduced tRFP657-co-expressing non-targeting or Vcam1targeting sgRNAs into mouse MLL-AF9/Cas9 cells and transplanted these cells into recipient mice (Fig 6I). Vcam1 depletion promoted the leukemic progression of MLL-AF9 cells, as evidenced by the increase of Vcam1-depleted (tRFP657 + ) cells in the bone marrow of the mice (Fig 6J). Taken together, these data suggest that Vcam1 promotes excessive cell-cell interaction of AML cells, which prevents their efficient proliferation both in vitro and in vivo.

Co-treatment with IMPDH inhibitors and TLR1/2 agonist shows strong antileukemia effects
Because the TLR1/TLR2 agonist Pam3CSK4 was shown to inhibit MLL-AF9-driven leukemogenesis (Eriksson et al, 2017), we next assessed the combined therapeutic effect of MPA and Pam3CSK4 on human MLL-AF9-expressing CB cells and the in vivo mouse AML model driven by MLL-AF9. Consistent with earlier results and the previous report (Eriksson et al, 2017), both MPA and Pam3CSK4 induced upregulation of myeloid markers CD11b and CD14 in MLL- AF9 cells in vitro, and cotreatment with these drugs strongly promoted differentiation of MLL-AF9 cells toward macrophage (Fig 7A  and B). Furthermore, cotreatment with Pam3CSK4 and MPA-induced Vcam1 upregulation in MLL-AF9 cells, which probably contributes to their antileukemia effect (Fig 7C). We then assessed the in vivo effects of FF-10501-01 (160 mg/kg) and Pam3CSK4 (8 lg/mouse) on MLL-AF9-induced leukemogenesis (Fig 7D). Although Pam3CSK4 alone did not inhibit the leukemic progression of MLL-AF9 cells, combined treatment of FF-10501-01 and Pam3CSK4 significantly prolonged the survival of leukemia mice than treatment with either FF-10501-01 or Pam3CSK4 alone (Fig 7E  and F). These findings suggest that co-treatment with IMPDH inhibitors and TLR1/2 agonist could be a promising therapeutic strategy for MLL-fusion AMLs.

Discussion
Although IMPDH has been considered as a potential anticancer drug target, the therapeutic effect of IMPDH inhibitors has not been proven in the clinical setting. It is therefore important to determine the types of tumors that are susceptible to IMPDH inhibitors and to establish optimal dose and treatment schedule to maximize their effects. In this study, we identified MLL-fusion leukemias as sensitive tumors to IMPDH inhibition. Furthermore, we showed that alternate-day administration of MPA and FF-10501-01 to mice effectively suppressed MLL-AF9-driven leukemogenesis without having a devastating effect on immune function. The clinical activity of IMPDH inhibitors to MLL-fusion leukemias warrants further investigation in clinical trials. Mechanisms underlying the anticancer activity of IMPDH inhibitors have been investigated for many years. Although it has been shown that IMPDH inhibition activates the p53-p21 pathway (Messina et al, 2004;Sun et al, 2008), our current and previous ) data indicate that this pathway is not essential for the anticancer effect of IMPDH inhibitors. Instead, we found that IMPDH inhibitors provoke overactivation of TLR-TRAF6-NF-jB signaling, which induces differentiation of AML cells. Because the MPA-induced activation of TLR-TRAF6-NF-jB signaling was reversed by guanosine supplementation, the reduced level of guanine nucleotides is likely to be triggers for the activation of this pathway. Given that most MLL-fusion AMLs have monocytic and macrophage-like characteristics and the TLR signaling plays key roles in monocytes/macrophages, the active TLR signaling in MLLfusion AMLs could explain why they are sensitive to IMPDH inhibitors. We also found the strong antileukemia effects of the combined treatment with IMPDH inhibitors and TLR1/2 agonist on MLL-AF9driven AML. Taken together, we propose that tumors with active TLR signaling, not limited to MLL-fusion AMLs, would respond well to IMPDH inhibitors. Furthermore, given the importance of TLR signaling in innate immune cells, our findings raise the possibility that IMPDH may have an important role in regulating the balance between innate and adaptive immunity. These hypotheses need to be verified in future research.
VCAM1 is a key cell adhesion molecule involved in inflammation (Kong et al, 2018). Although VCAM1 expression is low in most primary AML cells and AML cell lines, IMPDH inhibition induced Vcam1 upregulation in MLL-AF9-AML cells. Moreover, our functional analyses revealed the critical role of Vcam1 to regulate cellcell interaction among AML cells, which prevents their leukemic proliferation. These data suggest that Vcam1 acts as a tumor suppressor and contributes to the antileukemia effect of IMPDH inhibitors on MLL-fusion AML. In contrast to our findings, Pinho et al (2022) recently showed that VCAM1 confers innate immune tolerance on leukemia stem cells, thereby promoting leukemogenesis driven by MLL-AF9 (Pinho et al, 2022). The seemingly contradictory observations could be explained by the differences in the timing to induce Vcam1 depletion and the context of major histocompatibility complex (MHC) class-I presentation. We induced Vcam1 depletion in established MLL-AF9 cells and used syngeneic mice as recipients, while Pinho et al (2022) expressed MLL-AF9 in bone marrow cells derived from Vcam1-deficient mice and used MHC-mismatched mice as recipients. Thus, it appears that Vcam1 has diverse and context-dependent roles in the initiation, progression, and maintenance of AML, which remains to be clarified in future studies.
The antileukemia effect of IMPDH inhibitors could be augmented by the combination with other drugs. We previously showed that azacitidine-resistant leukemia cells that acquire resistance to a hypomethylating agent Azacitidine were still sensitive to IMPDH inhibitors (Murase et al, 2016), indicating that they could be combined with hypomethylating agents to treat myeloid neoplasms. However, cotreatment with MPA and decitabine did not show synergistic effects on human CB cells expressing MLL-AF9. Instead, we found that MPA synergized with a BCL2 inhibitor (Venetoclax) to inhibit the growth of MLL-AF9 cells (Appendix Fig S3). Thus, combined treatment with IMPDH and BCL2 inhibitors could be promising frontline therapies for MLL-fusion leukemia.
In summary, we show the therapeutic potential of IMPDH inhibitors for MLL-fusion leukemia. Alternate-day administration of IMPDH inhibitors to mice suppresses the development of MLL-AF9driven AML in vivo without deteriorating the immune system. The in vitro and in vivo efficacy strongly support future research to maximize the impact of IMPDH inhibitors on MLL-fusion AML and potentially other hematopoietic neoplasms. Statistical significance was calculated by the log-rank test.

Human cord blood cells and patient samples
Data information: All data are shown as mean AE SEM.
according to an institutional review board-approved protocol (approval number: 27-34-1225), in accordance with the Declaration of Helsinki and The Belmont Report. Residual diagnostic specimens from AML or ALL patients at Cincinnati Children's Hospital Medical Center (CCHMC) were treated with OKT3 antibody and engrafted into NSG or NSGS mice (Wunderlich et al, 2014). The engrafted human AML and ALL cells were then collected from the bone marrow of the recipient mice and used for the experiments.

Mice and animal procedures
C57BL/6JJmsSlc mice were purchased from Japan SLC, Inc. and used for experiments at the age of 8-12 weeks. Ly5.1 mice were maintained in IRCMS, Kumamoto University. NOD.Cg-Rag1 tm1Mom Il2rg tm1Wjl /SzJ (NRG), NOD.Cg-Rag1tm1MomIl2rgtm1WjlTg (CMV-IL3, CSF2, KITLG)1Eav/J (NRGS) and NOD.Cg-Prkdcscid Il2rgtm WjlTg (CMV-IL3, CSF2, KITLG)1Eav/MloySzJ (NSGS) mice were bred and maintained in the pathogen-free facility of CCHMC. The p53 À/À mice, in which 5 0 part of exon 2 including translation initiation site of Trp53 gene was replaced with Neomycin resistance gene, were provided from the RIKEN BioResource Center (Ibaragi, Japan; Tsukada et al, 1993). All animal experiments were performed in accordance with approved protocols from the Laboratory Animal Research Center of The Institute of Medical Science at the University of Tokyo (approval number: PA15-109, PA18-46) and with an approved animal study IACUC protocol at CCHMC.

In vitro drug sensitivity assay
To test the sensitivity of AML cells to IMPDH inhibitors, cells were cultured in a 96-well flat-bottom plate at a density of 2-5 × 10 4 /well in 100 ll of culture medium. Then, the cells were cultured with titrating doses of MPA (MP Biomedicals, Cat#194172; CAS:24280-93-1) or FF-10501-01 (FUJIFILM Corporation) together with or without 100 lM Guanosine (#G6264-6G, Sigma-Aldrich or ACROS ORGANICS, Cat#411130250; CAS:118-00-3). After 72 h, we added 10 ll of the WST-8 (#07553-44, Nacalai Tesque) or WST-1 (#11644807001, Roche) to each well and incubated the cells for 3-4 h. The absorbance of each sample was measured at a wavelength of 450 nm by a microplate reader. The combined effect of MPA + Decitabine and MPA + Venetoclax was evaluated with the similar in vitro assays. Combination index (CI) was calculated from the CI equation algorithms (Chou, 2006) using CompuSyn software.

Mouse transplantation assay and in vivo drug treatment
Mouse bone marrow c-Kit+ cells derived from wild-type or Trp53 (À/À) mice were transduced with MLL-AF9 (co-expressinf GFP) and were transplanted intravenously into sublethally irradiated (525 cGy) recipient mice. The MLL-AF9-expressing leukemia cells were then harvested from spleens of moribund mice and were serially transplanted into recipient mice. The serial transplantation was subsequently repeated several times to generate MLL-AF9 cells with strong leukemogenicity. These wild-type and Trp53(À/À) MLL-AF9 cells were injected intravenously (1 × 10 6 cells/mouse) into nonirradiated recipient mice. For drug studies, MMF (CAS RN: 128794-94-5, Product Number: M2387 from Tokyo chemical industry CO., Ltd), FF-10501-01 and DS-5272 (Daiichi Sankyo) were dissolved in 0.5 w/v% Methyl Cellulose 400 Solution (#133-17815, Wako, Japan). Pam3CSK4 (Catalog Code: tlrl-pms, InvivoGen) was dissolved in PBS. The MLL-AF9-bearing mice were orally administered with vehicle or the IMPDH inhibitors (120 mg/kg MMF and 160 mg/kg FF-10501-01) on an every 2-day schedule from Day 1 or Day 12 (FF-10501-01 and Pam3CSK4 combined treatments were started from Day 3) after the transplantation. In some experiments, the MLL-AF9-bearing mice were also intraperitoneally injected with 8 lg Pam3CSK4, or orally administered with 80 mg/kg of DS-5272. For the RNA-seq, flow cytometry analysis, single-cell mass cytometry, and metabolome analysis, the MLL-AF9-bearing mice were treated with 120 mg/kg MMF, 160 mg/kg FF-10501-01, or vehicle in two consecutive days. Bone marrow cells were then harvested from the mice 48 h after the first administration of the drugs.

Transfection and immunoprecipitation
Two hundred and ninety three T cells were transiently transfected with 3 lg of vector, FLAG-tagged TRAF6, and HA-tagged ubiquitin mixed with 30 ll polyethyleneimine (PEI). The cells were cultured for 48 h after the transfection and lysed in Cell Lysis Buffer (Cell Signaling Technology, Danvers, MA; #9803). For immunoprecipitation, the cell lysates were incubated with an anti-FLAG (SIGMA, #F3165) antibody for 30 min at 4°C. Then, the samples were incubated with Dynabeads protein-G (Themo Fisher Scientific, USA) for 30 min at 4°C. The precipitates were washed three times with the cell lysis buffer containing 1 mM phenylmethanesulfonyl fluoride, subjected to SDS/PAGE, and were analyzed by Western blotting with anti-HA (Roche Applied Sciences, 3F10, #11867423001) antibody.

Morphological analysis
Cytospin preparations were stained with Wright-Giemsa. Images were obtained with a BX51 microscope and a DP12 camera (Olympus).

Flow cytometry analysis
Mouse bone marrow cells were isolated from femurs and tibias using bone-crushing technique. After filtered into Falcon Cell Strainer (mesh size 40 lm, Cat# 087711), red cells were removed using 1x red blood cell (RBC) lysis Buffer. Cells were stained by antibodies (Appendix Table S3) in PBS containing 2% FBS (staining medium) for 30 min on ice. Cells were then washed twice in a staining medium and analyzed by FACS Verse or sorted by FACS Aria (BD Biosciences, San Jose, CA, USA). Cell cycle (#V35003, Vybrant DyeCycle Violet stain; Invitrogen) and apoptosis (Annexin V-APC kit; BD Biosciences) analyses were performed according to the manufacturer's recommendations.

Single-cell mass cytometry analysis
A summary of all mass cytometry antibodies, reporter isotopes, and concentrations used for analysis are provided in Appendix Table S4. Primary conjugates of mass cytometry antibodies were purchased preconjugated from Fluidigm or prepared using the MaxPAR antibody conjugation kit (Fluidigm PRD002 Version7) according to the manufacturer's recommended protocol. For sample preparation, MLL-AF9 cells were isolated from femurs and tibias of mice treated with vehicle (0.5 w/v% Methyl Cellulose 400 Solution) or FF-10501-01 24 h before. After filtration with Falcon Cell Strainer (mesh size 40 lm, Cat# 087711), red cells were removed using 1x Ammonium-Chloride-Potassium (ACK; Thermo Fisher, #A1049201) buffer. These cells were counted and resuspended in PBS (1 × 10 7 / ml), stained with live/dead cell indicator Cell-IDTM Cisplatin (Fluidigm, Cat# 201198) at a final concentration of 5 lM, and were then fixed with 1 × Maxpar FixI Buffer (Fluidigm, Cat# 201065). 1 × 10 6 cells/sample were aliquoted and were stained with the 26 antibodies (Appendix Table S4 (Fluidigm, Cat#107002). Approximately 100 K events per sample were acquired for each sample. FCS files were normalized to the EQ Four Element Calibration Beads using Helios software (Version 6.5.358) and were analyzed by SPADE. Gating and extraction of median expression levels were performed using Cytobank (https://premium.cytobank.org) under a condition of Target Number of Nodes is 150. The file was downsampled to an absolute number of 5,000. The CD45.2 + population and eight clustering channels (141Pr_Gr1,143rd_CD41,145_Nd_CD4,160Gd_B220,168Er_CD8a,170Er_CD49d,173Yb_ckit) were selected to create categorization for the SPADE.

Metabolome analysis
Mouse MLL-AF9 cells (1 × 10 6 cells/mouse) were intravenously injected into C57BL/6J recipient mice (total n = 6). MLL-AF9 mice were treated with vehicle or FF-10501-01 (160 mg/kg) 10 days after transplantation. Bone marrow cells were harvested from the mice 24 h after the treatment, and 5 × 10 5 GFP + cells were sorted for metabolome analysis. After metabolite extraction from sorted cells, metabolome analysis was performed as described previously (Kunisawa et al, 2015). Briefly, frozen sorted cell fractions together with internal standard (IS) compound 2-morpholinoethanesulfonic acid was suspended in ice-cold methanol (500 ll) followed by the addition of an equal volume of chloroform and 0.4 times the volume of ultrapure water (LC/MS grade, Wako). The suspension was then centrifuged at 15,000 g for 15 min at 4°C. After centrifugation, the aqueous phase was ultra-filtered using an ultrafiltration tube (Ultrafree MC-PLHCC, Human Metabolome Technologies). The filtrate was concentrated with a vacuum concentrator (SpeedVac, Thermo). The concentrated filtrate was dissolved in 25 ll of ultrapure water and used for ion chromatography (IC)-MS analyses as described below. For metabolome analysis of anion metabolites were measured using an orbitrap-type MS (Q-Exactive focus, Thermo Fisher Scientific, San Jose, CA), connected to a high-performance IC system (ICS-5000+, Thermo Fisher Scientific) that enables us to perform highly selective and sensitive metabolite quantification owing to the IC-separation and Fourier Transfer MS principle. The IC was equipped with an anion electrolytic suppressor (Thermo Scientific Dionex AERS 500) to convert the potassium hydroxide gradient into pure water before the sample enters the mass spectrometer. The separation was performed using a Thermo Scientific Dionex IonPac AS11-HC, 4-lm particle size column. IC flow rate was 0.25 ml/min supplemented post-column with 0.18 ml/min makeup flow of MeOH. The potassium hydroxide gradient conditions for IC separation are as follows: from 1 mM to 100 mM (0-40 min), 100 mM (40-50 min), and 1 mM (50.1-60 min), at a column temperature of 30°C. The Q Exactive focus mass spectrometer was operated under an ESI negative mode for all detections. Full mass scan (m/z 70-900) was used at a resolution of 70,000. The automatic gain control target was set at 3 × 10 6 ions, and maximum ion injection time (IT) was 100 ms. Source ionization parameters were optimized with the spray voltage at 3 kV and other parameters were as follows: transfer temperature at 320°C, S-Lens level at 50, heater temperature at 300°C, Sheath gas at 36, and Aux gas at 10.

RNA-Seq analysis
Mouse MLL-AF9 cells (1 × 10 6 cells/mouse) were intravenously injected into C57BL/6J recipient mice (total n = 6). MLL-AF9 mice were treated with vehicle or FF-10501-01 (160 mg/kg) 10 days after transplantation. Bone marrow cells were harvested from the mice 24 h after the treatment, and 5 × 10 5 GFP + cells were sorted for RNA-Seq. Total RNA was extracted using RNeasy Mini Kit (Qiagen), and the quality and quantity of RNA were checked using Agilent High Sensitivity RNA Screen Tape and Qubit. RNA libraries were prepared using 500 ng total RNA with SureSelect Strand-Specific RNA Preparation Kit (Agilent) according to the manufacturer's protocol. The quality and quantity of these libraries were checked using Agilent TapeStation D1000 and KAPA Library Quantification Kits [KAPA BioSystems] / Real-time PCR Systems Step One Plus [Applied Biosystems]. These libraries were sequenced on the Illumina HiSeq2500 System with 2 × 100 nucleotide paired end reads according to the manufacturer's protocol. Derived reads were processed using cutadapt (1.8.1) and fastx-toolkit (0.0.13) to remove Illumina adaptor sequence and to trim low-quality bases. Quality of reads were assessed using FastQC. Processed reads were aligned to GRCm38 reference transcripts using TopHat(2.1.1)-Cufflinks(2.2.1) pipeline (Trapnell et al, 2010(Trapnell et al, , 2014Kim et al, 2013) to derive gene FPKM values. For clustering analysis, normalized read counts were further transformed by the variance-stabilizing transformation method in DESeq2. They were then subjected to hierarchical clustering analysis with Ward's method.

Statistical analyses
GraphPad Prism 9 was used for statistical analyses. Unpaired Student's t-test (two-tailed) and Ordinary one-way ANOVA were used for pairwise comparisons of significance. The log-rank (Mantel-Cox) was used for the survival curves comparison. The differentially expressed gene of RNA-seq was analyzed by Cuffdiff. A Pvalue > 0.05 was considered as not significant (ns). Animal experiments were neither blinded nor randomized. The type of replication (biological or technical) is indicated in figure legends. Sample size was decided based on our previous experience in the field, not predetermined by a statistical method. All data are shown as mean AE SEM.

Data availability
All data needed to evaluate the conclusions are present in the paper and/or the Appendix files.
The datasets produced in this study are available in the following databases:

Results
Inosine monophosphate dehydrogenase (IMPDH) is an enzyme that play an essential role in guanine nucleotide synthesis pathway. IMPDH inhibitors have been prescribed to prevent rejection after organ transplantation. In this study, we found the potent antileukemia effect of two IMPDH inhibitors, MPA and FF-10501-01, on MLLfusion AMLs. The alternate-day administration of IMPDH inhibitors to mice effectively suppressed the development of AML in vivo without showing devastating effects on immune cells. As a mechanism, we found that IMPDH inhibitors induce overactivation of the TLR signaling and upregulation of VCAM1 in AML cells.

Impact
These data provide a rational basis for clinical testing of IMPDH inhibitors against MLL-fusion AMLs. Given the established safety as immunosuppressants, repurposing MPDH inhibitors could provide an affordable, safe, and effective therapy for leukemia patients. Our study also revealed the previously unrecognized crosstalk between intracellular levels of guanine nucleotides and TLR signaling, which will merit future studies.  The tRFP657 + cells were sorted and subjected to western blotting.

Expanded View Figures
Source data are available online for this figure. A B Figure EV3. Therapeutic effect of IMPDH inhibitor is significant on overt leukemia in mice.
A Experimental scheme used in (B). C57BL/6J mice were transplanted with mouse MLL-AF9-GFP cells and were treated with vehicle or FF-10501-01 from day 12 to 23, as indicated. GFP + MLL-AF9 leukemia cells collected from vehicle-or FF-10501-01-treated mice were analyzed on day 13. B (left) Kaplan-Meier survival curves of MLL-AF9 leukemia mice treated with vehicle or MMF (n = 11 per group). Statistical significance was evaluated by the log-rank test. (right) Frequencies of GFP + leukemic cells in peripheral blood at day 13 are also shown (vehicle: n = 3, FF-10501-01: n = 5). A two-tailed unpaired t-test was used for the comparison. Data are shown as mean AE SEM.
Source data are available online for this figure. Source data are available online for this figure.